Desaturases are enzymes involved in the synthesis of long chain polyunsaturated fatty acids (PUFAs). PUFAs are fatty acids (FAs) which are essential to the normal functioning of a cell and their nutritional properties are well known. An example of a PUFA is docosahexanoic acid (DHA). DHA is a n-3 fatty acid that can be obtained directly from the diet or derived from metabolism of dietary linoleic and α-linolenic acid. The n-3 fatty acids are associated with health promoting properties. For example n-3 fatty acids have been described as anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic and vasodilatory. As such, the role of DHA in the prevention and/or treatment of diseases such as coronary heart disease, hypertension, type II diabetes, ocular diseases, arthritis, cystic fibrosis and schizophrenia has been the focus of a great deal of medical research.
The production of PUFAs involves a consecutive series of desaturations and elongations of the fatty acyl chain to generate arachidonic acid (20:4Δ5,8,11,14) and docosahexaenoic acid (22:6Δ4,7,10,13,16,19). Several desaturases involved in this metabolic process have been isolated from marine microalgae, including Phaeodactylum tricornutum [5], Euglena gracilis [6] and Pavlova lutheri [7]. These membrane-bound desaturases are specific with respect to both chain length of the substrate and the double bond positions on the fatty acid. They belong to the class known as front-end fatty acid desaturases due to the fact that they introduce double bonds between the carboxy-group and pre-existing bond(s) of the fatty acid [1]. These desaturases contain a cytochrome b5 domain at their N-terminus and three histidine motifs that are important for catalytic activity [10].
Desaturase enzymes and the genes which encode them are known in the art. For example, WO03/064596 describes, amongst other things, transgenic cells transformed with omega 3 and delta 12 desaturase nucleic acid molecules and the use of these cells in the production of fatty acids. In particular the use of the omega 3 desaturase in the conversion of arachidonic acid to eicosapentaenoic acid and the use of the delta 12 desaturase in the conversion of oleic acid to linoleic acid. WO03/099216 also describes fungal desaturases and in particular transgenic plants modified to express fungal delta 15 desaturase enzymes.
Furthermore, US2003/0157144 and US2003/0167525 disclose delta 5 and delta 6 desaturase genes in the conversion of dihomoylinolenic acid to arachidonic acid and linoleic acid to y-linolenic acid respectively. Moreover, US2003/134400 discloses delta 4 desaturase genes which are involved in the conversion of adrenic acid to ω6-docosapentaenoic acid and in the conversion of ω3-docosapentaenoic acid to docosahexaenoic acid. These rare fatty acids are used in pharmaceutical and cosmetic compositions and can be essential nutritional fatty acids.
Besides the common FAs 16:0, 16:1Δ9, 18:0 and 18:1Δ9 found in most living organisms, trace amounts of more unusual fatty acids can be found in a wide range of species. For instance, presence of 16:1Δ11 has been reported in several species of Pavlova, in the Eustigmatophyte Nannochloropsis oculata, and in the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana [11,12,13]. This FA accounted for a very small portion of the total FAs in these microalgae, and its specific role in the algal cells is unknown. However, this FA is a very important precursor in the synthesis of sex pheromones in insects. Sex pheromones are species-specific blends of unsaturated fatty acid (UFA) derivatives that differ in terminal functional group and in the number, position and configuration (Z or E) of the double bond(s), which are produced by various acyl-CoA desaturases [14,15]. Simple monoene Δ11 UFAs are the most prevalent precursors in the formation of major sex pheromone components in the modern Lepidoptera [16,17]. For instance, in the corn earworm Helicoverpa zea, which produces a pheromone mixture of Z11-16:Ald and Z9-16:Ald in a 30:1 ratio, the most abundant desaturase-encoding transcript is HzeaLPAQ (also called HzPGDs1) which encodes a Δ11-desaturase that does not possess a cytochrome b5 extension, and therefore requires free cytochrome b5 for activity. Many acyl-CoA Δ11-desaturases with different specificities have been isolated from insects [14,15], but none from other species.